Treatment for diabetes

ABSTRACT

Methods and compositions for treating diabetes mellitus in a patient in need thereof are provided. The methods include administering to a patient a composition providing a gastrin/CCK receptor ligand, e.g., a gastrin, and/or an epidermal growth factor (EGF) receptor ligand, e.g., TGF-α, in an amount sufficient to effect differentiation of pancreatic islet precursor cells to mature insulin-secreting cells. The composition can be administered systemically or expressed in situ by cells transgenically supplemented with one or both of a gastrin/CCK receptor ligand gene, e.g., a preprogastrin peptide precursor gene and an EGF receptor ligand gene, e.g., a TGF-α gene. The methods also include transplanting into a patient cultured pancreatic islets in which mature insulin-secreting beta cells are proliferated by exposure to a gastrin/CCK receptor ligand and an EGF receptor ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.09/127,028, filed Jul. 30, 1998, which is a continuation U.S. Ser. No.07/992,255, filed Dec. 14, 1992, which issued Mar. 23, 1999, as U.S.Pat. No. ______, which disclosures are incorporated herein by reference.

INTRODUCTION

[0002] 1. Field of Invention

[0003] This invention relates to a method for treating diabetes mellitusin an individual in need thereof by administering to the individual acomposition comprising a gastrin/CCK receptor ligand and/or an EGFreceptor ligand which effectively promotes differentiation of pancreaticislet precursor cells to mature insulin-secreting cells. The method isexemplified by administration of gastrin and transforming growth factoralpha (TGF-α) either alone or in combination to normal streptozotocin(STZ) induced diabetic and genetically predisposed diabetic Zucker rats.

[0004] 2. Background

[0005] Diabetes is one of the most common endocrine diseases across allage groups and populations. In addition to the clinical morbidity andmortality, the economic cost of diabetes is huge, exceeding $90 billionper year in the US alone, and the prevalence of diabetes is expected toincrease more than two-fold by the year 2010.

[0006] There are two major forms of diabetes mellitus: insulin-dependent(Type 1) diabetes mellitus (IDDM) which accounts for 5 to 10% of allcases, and non-insulin dependent (Type 2) diabetes mellitus (NIDDM)which comprises roughly 90% of cases. Type 2 diabetes is associated withincreasing age however there is a trend of increasing numbers of youngpeople diagnosed with NIDDM, so-called maturity onset diabetes of theyoung (MODY). In both Type 1 and Type 2 cases, there is a loss ofinsulin secretion, either through destruction of the β-cells in thepancreas or defective secretion or production of insulin. In NIDDM,patients typically begin therapy by following a regimen of an optimaldiet, weight reduction and exercise. Drug therapy is initiated whenthese measures no longer provide adequate metabolic control. Initialdrug therapy includes sulfonylureas that stimulate β-cell insulinsecretion, but also can include biguanides, α-glucosidase inhibitors,thiazolidenediones and combination therapy. It is noteworthy howeverthat the progressive nature of the disease mechanisms operating in type2 diabetes are difficult to control. Over 50% of all drug-treateddiabetics demonstrate poor glycemic control within six years,irrespective of the drug administered. Insulin therapy is regarded bymany as the last resort in the treatment of Type 2 diabetes, and thereis patient resistance to the use of insulin.

[0007] Pancreatic islets develop from endodermal stem cells that lie inthe fetal ductular pancreatic endothelium, which also containspluripotent stem cells that develop into the exocrine pancreas.Teitelman and Lee, Developmental Biology, 121:454466 (1987); Pictet andRutter, Development of the embryonic encocrine pancreas, inEndocrinology, Handbook of Physiology, ed. R. O. Greep and E. B. Astwood(1972), American Physiological Society: Washington, D.C., p.25-66. Isletdevelopment proceeds through discrete developmental stages during fetalgestation which are punctuated by dramatic transitions. The initialperiod is a protodifferentiated state which is characterized by thecommitment of the pluripotent stem cells to the islet cell lineage, asmanifested by the expression of insulin and glucagon by theprotodifferentiated cells. These protodifferentiated cells comprise apopulation of committed islet precursor cells which express only lowlevels of islet specific gene products and lack the cytodifferentiationof mature islet cells. Pictet and Rutter, supra. Around day 16 in mousegestation, the protodifferentiated pancreas begins a phase of rapidgrowth and differentiation characterized by cytodifferentiation of isletcells and a several hundred fold increase in islet specific geneexpression. Histologically, islet formation (neogenesis) becomesapparent as proliferating islets bud from the pancreatic ducts(nesidioblastosis). Just before birth the rate of islet growth slows,and islet neogenesis and nesidioblastosis becomes much less apparent.Concomitant with this, the islets attain a fully differentiated statewith maximal levels of insulin gene expression. Therefore, similar tomany organs, the completion of cellular differentiation is associatedwith reduced regenerative potential; the differentiated adult pancreasdoes not have either the same regenerative potential or proliferativecapacity as the developing pancreas.

[0008] Since differentiation of protodifferentiated precursors occursduring late fetal development of the pancreas, the factors regulatingislet differentiation are likely to be expressed in the pancreas duringthis period. One of the genes expressed during islet development encodesthe gastrointestinal peptide, gastrin. Although gastrin acts in theadult as a gastric hormone regulating acid secretion, the major site ofgastrin expression in the fetus is the pancreatic islets. Brand andFuller, J. Biol Chem., 263:5341-5347 (1988). Expression of gastrin inthe pancreatic islets is transient. It is confined to the period whenprotodifferentiated islet precursors form differentiated islets.Although the significance of pancreatic gastrin in islet development isunknown, some clinical observations suggest a rule for gastrin in thisislet development as follows. For example, hypergastrinemia caused bygastrin-expressing islet cell tumors and atrophic gastritis isassociated with nesidioblastosis similar to that seen in differentiatingfetal islets. Sacchi, et al., Virchows Archiv B, 48:261-276 (1985); andHeitz et al., Diabetes, 26:632-642 (1977). Further, an abnormalpersistence of pancreatic gastrin has been documented in a case ofinfantile nesidioblastosis. Hollande, et al., Gastroenterology,71:251-262 (1976). However, in neither observation was a causalrelationship established between the nesidioblastosis and gastrinstimulation.

[0009] It is therefore of interest to identify agents that stimulateislet cell regeneration which could be of value in the treatment ofearly IDDM and in the prevention of β-cell deficiency in NIDDM.

[0010] Citations of a reference herein shall not be construed as anadmission that such reference is prior art to the present invention.

RELEVANT LITERATURE

[0011] Three growth factors are implicated in the development of thefetal pancreas, gastrin, transforming growth factor a (TGF-α) andepidermal growth factor (EGF) (Brand and Fuller, J. Biol Chem.263:5341-5347). Transgenic mice over expressing TGF-α or gastrin alonedid not demonstrate active islet cell growth, however nice expressingboth transgenes displayed significantly increased islet cell mass (Wanget al, (1993) J Clin Invest 92:1349-1356). Bouwens and Pipeleers (1998)Diabetoligia 41:629-633 report that there is a high proportion ofbudding β-cells in the normal adult human pancreas and 15% of allβ-cells were found as single units. Single β-cell foci are not commonlyseen in adult (unstimulated) rat pancreas; Wang et al ((1995)Diabetologia 38:1405-1411) report a frequency of approximately 1% oftotal β-cell number.

[0012] Insulin independence in a Type I diabetic patient afterencapsulated islet transplantation is described in Soon-Shiong et al(1994) Lancet 343:950-51. Also see Sasaki et al (1998 Jun 15)Transplantation 65(11): 1510-1512; Zhou et al (1998 May) Am J Physiol274(5 Pt 1):C1356-1362; Soon-Shiong et al (1990 June) Postgrad Med87(8):133-134; Kendall et al (1996 June) Diabetes Metab 22(3): 157-163;Sandler et al (1997 June) Transplantation 63(12):1712-1718; Suzuki et al(1998 January) Cell Transplant 7(1):47-52; Soon-Shiong et al (1993 June)Proc Natl Acad Sci USA 90(12):5843-5847; Soon-Shiong et al (1992November) Transplantation 54(5):769-774; Soon-Shiong et al (1992October) ASAIO J 38(4):851-854; Benhamou et al (1998 June) DiabetesMetab 24(3):215-224; Christiansen et al (1994 December) J ClinEndocrinol Metab 79(6):1561-1569; Fraga et al (1998 April)Transplantation 65(8):1060-1066; Korsgren et al (1993) Ups J Med Sci98(1):39-52; Newgard et al (1997 July) Diabetologiz 40 Suppl 2:S42-S47.

SUMMARY OF THE INVENTION

[0013] The invention provides methods for treating diabetes mellitus ina patient in need thereof by administering a composition providing agastrin/CCK receptor ligand, an EGF receptor ligand, or a combination ofboth in an amount sufficient to effect differentiation of the patient'spancreatic islet precursor cells to mature insulin-secreting cells. Thecomposition can be administered systemically or expressed in situ byhost cells containing a nucleic acid construct in an expression vectorwherein the nucleic acid construct comprises a coding sequence for agastrin CCK receptor ligand or a coding sequence for an EGF receptorligand, together with transcriptional and translational regulatoryregions functional in pancreatic islet precursor cells. Also providedare methods and compositions for treating diabetes in a patient in needthereof by implanting into a diabetic patient pancreatic islet cellsthat have been exposed in culture to a sufficient amount of agastrin/CCK receptor ligand and an EGF receptor ligand to increase thenumber of pancreatic beta cells in the islets; optionally the populationof pancreatic beta cells can be grown in culture for a time sufficientto expand the population of β-cells prior to transplantation. Themethods and compositions find use in treating patients with diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1A is an image that shows numerous insulin staining cells inthe metaplastic ducts from the TGF-α transgenic pancreas uponimmunoperoxidase staining. FIG. 1B is an image that shows that mostductular cells stained less intensely for insulin, while occasionalductular cells did stain with the same intensity of insulin staining asthe adjacent islets.

[0015]FIG. 2A schematically shows the structure of the chimeric insulinpromoter-gastrin (INSGAS) transgene. FIG. 2B illustrates that theradioimmunassay of pancreatic extracts from INSGAS transgenic mice showshigh levels of gastrin immunoreactivity that exceed the gastrin contentin the gastric antrum expressed from the endogenous murine gene. TheINSGAS transgenic mice had high expression of gastrin in the postnatalpancreas.

[0016]FIG. 3A is an image of the pancreatic histology of an INSGAS/TGF-αmouse used in the study reported by Example 3. The INSGAS/TGF-α pancreashad some areas of increased ductular complexes and slightly increasedinterstitial cellularity. The field shown here had the most severelyabnormal histology in the five animals used. FIG. 3B is an image of thepancreatic histology of a control mouse from Example 3. FIG. 3C is animage of the pancreatic histology of a TGF-α mouse from Example 3. Thisfield of a TGF-α mouse pancreas from the study reported in Example 3 wastypical and showed the interstitial cellularity and fibrosis combinedwith florid ductular metaplasia that has been described by Jhappan, etal, supra.

[0017]FIG. 4A is a histogram graphically illustrating point countingmorphometric data which confirmed that at 17 weeks the pancreas of theINSGAS/TGF-α mice had lower duct mass than the pancreas of the TGF-αmice based on the study reported in Example 3. FIG. 4B is a histogramwhich graphically illustrates point=counting morphometric data whichshow that co-expression of gastrin and TGF-α in the INSGAS/TGF-αpancreas significantly increased the islet mass compared to the isletmass of the corresponding non-transgenic control mice. Further, TGF-αexpression alone does not increase islet mass. These data are based onthe studies illustrated in Example 3.

[0018]FIG. 5 shows the effects of TGF-α and gastrin on glucose tolerancein streptozotocin induced diabetic Wistar rats treated with PBS (solidblack diamonds) or a combination of TGF-α and gastrin i.p. daily for 10days (solid purple squares).

[0019]FIG. 6 shows the effect of TGF-α and gastrin treatment on β-cellneogenesis in three groups of treated Zucker rats together with thecorresponding PBS controls (n=6 per group) as described in Example 7.The light blue bar represents lean TFG+gastrin, the magenta barrepresents ob TGF+gastrin, the yellow bar represents the ob PBS control,the dark blue bar represents pre TFG+gastrin and the purple barrepresents the lean PBS control. TGF-α and gastrin significantlyincreased the relative proportion of single β-cell foci in all thegroups studied as compared to PBS-treated control animals. Groups 4 and5 are significantly different (p<0.0015) as are Groups 1 and 2(p<0.0041).

[0020]FIG. 7 shows the effect of TGF-α and gastrin treatment on β-cellneogenesis in lean and obese Zucker rats. β-cell neogenesis isquantified by differential counting of total β-cells and newly generatedsingle β-cell foci and is expressed as a percentage of total β-cellscounted. The percentage of single β-cell foci in lean Zucker ratstreated with the growth factor combination was 10.5±0.9 compared to3.9±1.1 (p=0.004) in the corresponding PBS control (FIGS. 7A and 7B). Inthe obese Zucker rats, the percent single β-cell foci in thepretreatment group was 8.7±1.3 vs. 4.2±1.1 (p=0.0015) in thecorresponding control group (FIGS. 7C and 7D). FIG. 7E is a 400×magnification of the ductal region of FIG. 7C (indicated by an arrow)and provides clear evidence of the budding of insulin-containing β-cellsfrom the ductal epithelial cells characteristic of β-cell neogenesis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0021] The invention provides methods for treating diabetes mellitus ina patient in need thereof by administering a composition providing agastrin/CCK receptor ligand such as gastrin, an EGF receptor ligand,such as TGF-α, or a combination of both in an amount sufficient toeffect differentiation of pancreatic islet precursor cells to matureinsulin-secreting cells. When the composition is administeredsystemically, generally it is provided by injection, preferablyintravenously, in a physiologically acceptable carrier. When thecomposition is expressed in situ, pancreatic islet precursor cells aretransformed either in ex vivo or in vivo with one or more nucleic acidexpression constructs in an expression vector which provides forexpression of the desired receptor ligand(s) in the pancreatic isletprecursor cells. As an example, the expression construct includes acoding sequence for a CCK receptor ligand, such as preprogastrin peptideprecursor coding sequence which, following expression, is processed togastrin or a coding sequence for an EGF receptor ligand such as TGF-α,together with transcriptional and translational regulatory regions whichprovide for expression in the pancreatic islet precursor cells. Thetranscriptional regulatory region can be constitutive or induced, forexample by increasing intracellular glucose concentrations, such as atranscriptional regulatory region from an insulin gene. Transformationis carried out using any suitable expression vector, for example, anadenoviral expression vector. When the transformation is carried out exvivo, the transformed cells are implanted in the diabetic patient, forexample using a kidney capsule. Alternatively, pancreatic islet cellsare treated ex vivo with a sufficient amount of a gastrin/CCK receptorligand and/or an EGF receptor ligand to increase the number ofpancreatic β cells in the islets prior to implantation into the diabeticpatient. As required, the population of pancreatic 1 cells is expandedin culture prior to implantation by contacting them with the samereceptor ligand(s).

[0022] The subject invention offers advantages over existing treatmentregimens for diabetic patients. By providing a means to stimulate theadult pancreas to regenerate, not only is the need for traditional drugtherapy (Type 2) or insulin therapy (Type 1 and Type 2) reduced or eveneliminated, but the maintenance of normal blood glucose levels also mayreduce some of the more debilitating complications of diabetes. Diabeticcomplications include those affecting the small blood vessels in theretina, kidney, and nerves, (microvascular complications), and thoseaffecting the large blood vessels supplying the heart, brain, and lowerlimbs (mascrovascular complications). Diabetic microvascularcomplications are the leading cause of new blindness in people 20-74years old, and account for 35% of all new cases of end-stage renaldisease. Over 60% of diabetics are affected by neuropathy. Diabetesaccounts for 50% of all non-traumatic amputations in the USA, primarilyas a result of diabetic macrovascular disease, and diabetics have adeath rate from coronary artery disease that is 2.5 times that ofnon-diabetics. Hyperglycemia is believed to initiate and accelerateprogression of diabetic microvascular complications. Use of the variouscurrent treatment regimens cannot adequately control hyperglycemia andtherefore does not prevent or decrease progression of diabeticcomplications.

[0023] As used herein, the term “gastrin/CCK receptor ligand”encompasses compounds that stimulate the gastrin/CCK receptor. Examplesof such gastrin/CCK receptor ligands include various forms of gastrinsuch as gastrin 34 (big gastrin), gastrin 17 (little gastrin), andgastrin 8 (mini gastrin); various forms of cholecystokinin such as CCK58, CCK 33, CCK/22, CCK 12 and CCK 8; and other gastrin/CCK receptorligands that either alone or in combination with EGF receptor ligandscan induce differentiation of cells in mature pancreas to forminsulin-secreting islet cells. Also contemplated are active analogs,fragments and other modifications of the above. Such ligands alsoinclude compounds that increase the secretion of endogenous gastrins,cholecystokinins or similarly active peptides from sites of tissuestorage. Examples of these are omeprazole which inhibits gastric acidsecretion and soy bean trypsin inhibitor which increases CCKstimulation.

[0024] As used herein, the term “EGF receptor ligand” encompassescompounds that stimulate the EGF receptor such that when gastrin/CCKreceptors in the same or adjacent tissues or in the same individual alsoare stimulated, neogenesis of insulin-producing pancreatic islet cellsis induced. Examples of such EGF receptor ligands include EGF1-53, andfragments and active analogs thereof, including EGF1-48, EGF1-52,EGF1-49. See, for example, U.S. Pat. No. 5,434,135. Other examplesinclude TGF-α receptor ligands (1-50) and fragments and active analogsthereof, including 148, 147 and other EGF receptor ligands such asamphiregulin and pox virus growth factor as well as other EGF receptorligands that demonstrate the same synergistic activity with gastrin/CCKreceptor ligands. These include active analogs, fragments andmodifications of the above. For further background, see Carpenter andWahl, Chapter 4 in Peptide Growth Factors (Eds. Sporn and Roberts),Springer Verlag, (1990).

[0025] A principal aspect of the invention is a method for treatingdiabetes mellitus in an individual in need thereof by administering tothe individual a composition including a gastrin/CCK receptor ligandand/or an EGF receptor ligand in an amount sufficient to effectdifferentiation of pancreatic islet precursor cells to matureinsulin-secreting cells. The cells so differentiated are residual latentislet precursor cells in the pancreatic duct. One embodiment comprisesadministering, preferably systemically, a differentiation regenerativeamount of a gastrin/CCK receptor ligand and an EGF receptor ligand,preferably TGF-α, either alone or in combination to the individual.

[0026] Another embodiment comprises providing a gastrin/CCK receptorligand and/or an EGF receptor ligand to pancreatic islet precursor cellsof explanted pancreatic tissue of a mammal and reintroducing thepancreatic tissue so stimulated to the mammal.

[0027] In another, the invention comprises providing a gastrin/CCKreceptor ligand and/or an EGF receptor ligand to pancreatic isletprecursor cells of explanted pancreatic tissue from a mammal to expandthe population of β cells.

[0028] In another embodiment gastrin/CCK receptor ligand stimulation iseffected by expression of a chimeric insulin promoter-gastrin fusiongene construct transgenically introduced into such precursor cells. Inanother embodiment EGF receptor ligand stimulation is effected byexpression of an EGF receptor ligand gene transgenically introduced intothe mammal. The sequence of the EGF gene is provided in U.S. Pat. No.5,434,135.

[0029] In another embodiment stimulation by a gastrin/CCK receptorligand and an EGF receptor ligand is effected by coexpression of (i) apreprogastrin peptide precursor gene and (ii) an EGF receptor ligandgene that have been stably introduced into the mammal.

[0030] In another aspect the invention relates to a method for effectingthe differentiation of pancreatic islet precursor cells of a mammal bystimulating such cells with a combination of a gastrin/CCK receptorligand and an EGF receptor ligand. In a preferred embodiment of thisaspect, gastrin stimulation is effected by expression of a preprogastrinpeptide precursor gene stably introduced into the mammal. The expressionis under the control of the insulin promoter. EGF receptor iigand, e.g.,TGF-α, stimulation is effected by expression of an EGF receptor ligandgene transgenically introduced into the mammal. In furtherance of theabove, stimulation by a gastrin and a TGF-α is preferably effected byco-expression of (i) a preprogastrin peptide precursor gene and (ii) anEGF receptor ligand introduced into the mammal. Appropriate promoterscapable of directing transcription of the genes include both viralpromoters and cellular promoters. Viral promoters include the immediateearly cytomegalovirus (CMV) promoter (Boshart et al (1985) Cell41:521-530), the SV40 promoter (Subramani et al (1981) Mol. Cell. Biol.1:854-864) and the major late promoter from Adenovirus 2 (Kaufman andSharp (1982) Mol. Cell. Biol. 2:1304-13199). Preferably, expression ofone or both of the gastrin/CCK receptor ligand gene and the EGF receptorligand gene is under the control of an insulin promoter.

[0031] Another aspect of the invention is a nucleic acid construct. Thisconstruct includes a nucleic acid sequence coding for a preprogastrinpeptide precursor and an insulin transcriptional regulatory sequence,which is 5′ to and effective to support transcription of a sequenceencoding the preprogastrin peptide precursor. Preferably, the insulintranscriptional regulatory sequence includes at least an insulinpromoter. In a preferred embodiment the nucleic acid sequence coding forthe preprogastrin peptide precursor comprises a polynucleotide sequencecontaining exons 2 and 3 of a human gastrin gene and optionally alsoincluding introns 1 and 2.

[0032] Another embodiment of the invention is an expression cassettecomprising (i) a nucleic acid sequence coding for a mammalian EGFreceptor ligand, e.g., TGF-α and a transcriptional regulatory sequencethereof; and (ii) a nucleic acid sequence coding for the preprogastrinpeptide precursor and a transcriptional regulatory sequence thereof.Preferably, the transcriptional regulatory sequence for the EGF receptorligand is a strong non-tissue specific promoter, such as ametallothionein promoter. Preferably, the transcriptional regulatorysequence for the preprogastrin peptide precursor is an insulin promoter.A preferred form of this embodiment is one wherein the nucleic acidsequence coding for the preprogastrin peptide precursor comprises apolynucleotide sequence containing introns 1 and 2 and exons 2 and 3 ofthe human gastrin gene.

[0033] Another aspect of the invention relates to a vector including theexpression cassette comprising the preprogastrin peptide precursorcoding sequence. This vector can be a plasmid such as pGem1 or can be aphage which has a transcriptional regulatory sequence including aninsulin promoter.

[0034] Another aspect of this invention relates to a composition ofvectors including (1) having the nucleic acid sequence coding for amammalian EGF receptor ligand, e.g., TGF-α, under control of a strongnon-tissue specific promoter, e.g., a metallothionein promoter; and apreprogastrin peptide precursor coding sequence under control of aninsulin promoter. Each vector can be a plasmid, such as plasmid pGem1 ora phage in this aspect. Alternatively, the expression cassette or vectoralso can be inserted into a viral vector with the appropriate tissuetrophism. Examples of viral vectors include adenovirus, Herpes simplexvirus, adeno-associated virus, retrovirus, lentivirus, and the like. SeeBlomer et al (1996) Human Molecular Genetics 5 Spec. No:1397404; andRobbins et al (1998) Trends in Biotechnology 16:35-40.Adenovirus-mediated gene therapy has been used successfully totransiently correct the chloride transport defect in nasal epithelia ofpatients with cystic fibrosis. See Zabner et a. (1993) Cell 75:207-216.

[0035] Another aspect of the invention is a non-human mammal ormammalian tissue, including cells, thereof capable of expressing astably integrated gene which encodes preprogastrin. Another embodimentof this aspect is a non-human mammal capable of coexpressing (i) apreprogastrin peptide precursor gene; and/or (ii) an EGF receptorligand, e.g., a TGF-α gene that has been stably integrated into thenon-human mammal, mammalian tissue or cells. The mammalian tissue orcells can be human tissue or cells.

[0036] Therapeutic Administration and Compositions

[0037] Modes of administration include but are not limited totransdermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, and oral routes. The compounds can be administered by anyconvenient route, for example by infusion or bolus injection byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and can be administeredtogether with other biologically active agents. Administration ispreferably systemic.

[0038] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of atherapeutic, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Pharmaceuticallyacceptable carriers and formulations for use in the present inventionare found in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 17^(th) ed. (1985), which is incorporatedherein by reference. For a brief review of methods for drug delivery,see Langer (1990) Science 249:1527-1533, which is incorporated herein byreference.

[0039] In preparing pharmaceutical compositions of the presentinvention, it may be desirable to modify the compositions of the presentinvention to alter their pharmacokinetics and biodistribution. For ageneral discussion of pharmacokinetics, see Remingtons's PharmaceuticalSciences, supra, Chapters 37-39. A number of methods for alteringpharmacokinetics and biodistribution are known to one of ordinary skillin the art (See, e.g., Langer, supra). Examples of such methods includeprotection of the agents in vesicles composed of substances such asproteins, lipids (for example, liposomes), carbohydrates, or syntheticpolymers. For example, the agents of the present invention can beincorporated into liposomes in order to enhance their pharmacokineticsand biodistribution characteristics. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al (1980) Ann.Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and4,837,028, all of which are incorporated herein by reference. Variousother delivery systems are known and can be used to administer atherapeutic of the invention, e.g., microparticles, microcapsules andthe like.

[0040] The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulations can include standard carriers suchas pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, etc.

[0041] In a preferred embodiment, the composition is formulated inaccordance with routine procedures such as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition also caninclude a solubilizing agent and a local anesthetic to ameliorate anypain at the site of the injection. Generally, the ingredients aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quality of active agent. Where the composition is to be administeredby infusion, it can be dispensed with an infusion bottle containingsterile pharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

[0042] The therapeutics of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree amino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium and other divalent cations, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

[0043] The amount of the therapeutic of the invention which is effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. The precise dose to be employed in theformulation also will depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.However, suitable dosage ranges for intravenous administration aregenerally about 20-500 micrograms of active compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 μg/kg body weight to 1 mg/kg body weight. Effectivedosages can be extrapolated from dose-response curves derived from invitro or animal model test systems. Suppositories generally containactive ingredient in the range of 0.5% to 10% weight; oral formulationspreferably contain 10% to 95% active ingredient.

[0044] In the gene therapy methods of the invention, transfection invivo is obtained by introducing a therapeutic transcription orexpression vector into the mammalian host, either as naked DNA,complexed to lipid carriers, particularly cationic lipid carriers, orinserted into a viral vector, for example a recombinant adenovirus. Theintroduction into the mammalian host can be by any of several routes,including intravenous or intraperitoneal injection, intratracheally,intrathecally, parenterally, intraarticularly, intranasally,intramuscularly, topically, transdermally, application to any mucousmembrane surface, corneal installation, etc. Of particular interest isthe introduction of the therapeutic expression vector into a circulatingbodily fluid or into a body orifice or cavity. Thus, intravenousadministration and intrathecal administration are of particular interestsince the vector may be widely disseminated following such routes ofadministration, and aerosol administration finds use with introductioninto a body orifice or cavity. Particular cells and tissues can betargeted, depending upon the route of administration and the site ofadministration. For example, a tissue which is closest to the site ofinjection in the direction of blood flow can be transfected in theabsence of any specific targeting. If lipid carriers are used, they canbe modified to direct the complexes to particular types of cells usingsite-directing molecules. Thus, antibodies or ligands for particularreceptors or other cell surface proteins may be employed, with a targetcell associated with a particular surface protein.

[0045] Any physiologically acceptable medium may be employed foradministering the DNA, recombinant viral vectors or lipid carriers, suchas deionized water, saline, phosphate-buffered saline, 5% dextrose inwater, and the like as described above for the pharmaceuticalcomposition, depending upon the route of administration. Othercomponents can be included in the formulation such as buffers,stabilizers, biocides, etc. These components have found extensiveexemplification in the literature and need not be described inparticular here. Any diluent or components of diluents that would causeaggregation of the complexes should be avoided, including high salt,chelating agents, and the like.

[0046] The amount of therapeutic vector used will be an amountsufficient to provide for a therapeutic level of expression in a targettissue. A therapeutic level of expression is a sufficient amount ofexpression to decrease blood glucose towards normal levels. In addition,the dose of the nucleic acid vector used must be sufficient to produce adesired level of transgene expression in the affected tissues in vivo.Other DNA sequences, such as adenovirus VA genes can be included in theadministration medium and be co-transfected with the gene of interest.The presence of genes coding for the adenovirus VA gene product maysignificantly enhance the translation of mRNA transcribed from theexpression cassette if this is desired.

[0047] A number of factors can affect the amount of expression intransfected tissue and thus can be used to modify the level ofexpression to fit a particular purpose. Where a high level of expressionis desired, all factors can be optimized, where less expression isdesired, one or more parameters can be altered so that the desired levelof expression is attained. For example, if high expression would exceedthe therapeutic window, then less than optimum conditions can be used.

[0048] The level and tissues of expression of the recombinant gene maybe determined at the mRNA level as described above, and/or at the levelof polypeptide or protein. Gene product may be quantitated by measuringits biological activity in tissues. For example, protein activity can bemeasured by immunoassay as described above, by biological assay such asblood glucose, or by identifying the gene product in transfected cellsby immunostaining techniques such as probing with an antibody whichspecifically recognizes the gene product or a reporter gene productpresent in the expression cassette.

[0049] Typically, the therapeutic cassette is not integrated into thepatient's genome. If necessary, the treatment can be repeated on an adhoc basis depending upon the results achieved. If the treatment isrepeated, the patient can be monitored to ensure that there is noadverse immune or other response to the treatment.

[0050] The invention also provides for methods for expanding apopulation of pancreatic β-cells in vitro. Upon isolation of thepancreas from a suitable donor, cells are isolated and grown in vitro.The cells which are employed are obtained from tissue samples frommammalian donors including human cadavers, porcine fetuses or anothersuitable source of pancreatic cells. If human cells are used, whenpossible the cells are major histocompatability matched with therecipient. Purification of the cells can be accomplished by gradientseparation after enzymatic (e.g., collagenase) digestion of the isolatedpancreas.

[0051] The purified cells are grown in media containing sufficientnutrients to allow for survival of the cells as well as a sufficientamount of a β-cell proliferation inducing composition containing agastrin/CCK receptor ligand and EGF receptor ligand, to allow forformation of insulin secreting pancreatic β cells. According to theinvention, following stimulation the insulin secreting pancreatic βcells can be directly expanded in culture prior to being transplantedinto a patient in need thereof, or can be transplanted directlyfollowing treatment with β-cell proliferation inducing composition.

[0052] Methods of transplantation include transplanting insulinsecreting pancreatic β-cells obtained into a patient in need thereof incombination with immunosuppressive agents, such as cyclosporin. Theinsulin producing cells also can be encapsulated in a semi-permeablemembrane prior to transplantation. Such membranes permit insulinsecretion from the encapsulated cells while protecting the cells fromimmune attack. The number of cells to be transplanted is estimated to bebetween 10,000 and 20,000 insulin producing β cells per kg of thepatient Repeated transplants may be required as necessary to maintain aneffective therapeutic number of insulin secreting cells. The transplantrecipient can also, according to the invention, be provided with asufficient amount of a gastrin/CCK receptor ligand and an EGF receptorligand to induce proliferation of the transplanted insulin secreting βcells.

[0053] The effect of treatment of diabetes can be evaluated as follows.Both the biological efficacy of the treatment modality as well as theclinical efficacy are evaluated, if possible. For example, diseasemanifests itself by increased blood sugar, the biological efficacy ofthe treatment therefore can be evaluated, for example, by observation ofreturn of the evaluated blood glucose towards normal. The clinicalefficacy, i.e. whether treatment of the underlying effect is effectivein changing the course of disease, can be more difficult to measure.While the evaluation of the biological efficacy goes a long way as asurrogate endpoint for the clinical efficacy, it is not definitive.Thus, measuring a clinical endpoint which can give an indication ofβ-cell regeneration after, for example, a six-month period of time, cangive an indication of the clinical efficacy of the treatment regimen.

[0054] The subject compositions can be provided as kits for use in oneor more procedures. Kits for genetic therapy usually will include thetherapeutic DNA construct either as naked DNA with or withoutmitochondrial targeting sequence peptides, as a recombinant viral vectoror complexed to lipid carriers. Additionally, lipid carriers can beprovided in separate containers for complexing with the provided DNA.The kits include a composition comprising an effective agent either asconcentrates (including lyophilized compositions), which can be dilutedfurther prior to use or they can be provided at the concentration ofuse, where the vials may include one or more dosages. Conveniently, inthe kits single dosages can be provided in sterile vials so that thephysician can employ the vials directly, where the vials will have thedesired amount and concentration of agents. When the vials contain theformulation for direct use, usually there will be no need for otherreagents for use with the method. Associated with such kits can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

[0055] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0056] Materials and Methods

[0057] The following materials and methods were used in the studiesreported by the working examples set forth below except as otherwisenoted.

[0058] Animals. Mice, FVB and CD strain, were obtained from TaconicFarms, Inc., Germantown, N.Y. The TGF-α transgenic line MT-42 used,which expresses high levels of TGF-α from a metallothionein promoter, isdescribed in Jhappan et al, Cell, 61:1137-1146 (1990). Normal Wistar andZucker rats were allowed normal chow ad libidum with free access towater and were acclimatized for one week prior to initiation of eachstudy. Freshly prepared streptozotocin at a dose of 80 mg/kg body weightwas administered by I.V. five to seven days after induction of diabetes,the rats were randomly allocated into groups for subsequent treatment.Hormones, TGF-α and rat gastrin were reconstituted in sterile normalsaline containing 0.1% BSA. According to the predetermined treatmentschedule for different studies, each animal received a single, dailyi.p. injection of either TGF-α or gastrin alone (4.0 μg/kg body weight)or as a 1:1 (w/w) combination (total 8.0 μg/kg) or PBS for a period of10 days.

[0059] INSGAS Transgene Construct. A Pvull-Rsal fragment encompassingnucleotides −370 to +38 of the rat insulin I gene (Cordell, B. G. et al,Cell, 18:533-543 (1979)) was ligated into pGem1 (Promega Corp., Madison,Wis.). A 4.4 kb BamH1-EcoR1 fragment containing 1.5 kb introns 1 and 2and exons 2 and 3 of the human gastrin gene which encodes thepreprogastrin peptide precursor was isolated and subcloned downstream ofthe rat insulin I fragment in pGem1 (Promega). The fragment is describedin Wiborg, O., Proc. Natl. Acad. Sci. USA, 81:1067-1069 (1984) and Ito,R., et al Proc. Natl. Acada. Sci. (USA), 81:4662-4666 (1984). Theinsulin promoter-preprogastrin INSGAS transgene construct was excised asa 4.8 kb Xbal-EcoR1 fragment.

[0060] Generation and Characterization of Transgenic Mice. The fragment,made as described above was prepared for microinjection as follows. Itwas isolated by agarose gel electrophoresis, purified by CsCl gradientpurification, and dialyzed extensively against injection buffer (5 mMNaCl; 01. MM EDTA; 5 mM Tris-HCl pH 7.4). Fertilized oocytes from FVBinbred mice (Taconic Farms, Inc., supra) at the single-cell stage weremicroinjected using standard techniques. See Hogan, B., et al,Manipulating the mouse embryo: A laboratory manual, Cold Spring Harbor,N.Y. (1986). Surviving embryos were then implanted into the oviducts ofCD1 (Charles River Laboratories, Inc., Wilmington, Mass.) foster mothersaccording to procedures in Hogan et al. Transgenic founder mice wereidentified by DNA blot techniques using DNA isolated from individualmouse tails, and a human gastrin exon 2 probe labelled with 32 dCTP byrandom priming. F1 mice and their siblings were similarly identified.

[0061] Homozygous MT-42 mice containing the MT-TGF-α transgene derivedfrom a CD-1 mouse strain (Jhappan, supra) were crossed withheterozygotic INSGAS mice. After weaning, the offspring were placed onacidified 50 mM ZnCl₂ as previously described in order to induce themetallothionein promoter (Jhappan, supra).

[0062] Northern Blot Hybridization Assay. For Northern analysis, totalRNA was extracted from tissues by the method of Cathala et al, DNA2:329-335 (1983). Samples of 20 μg of total RNA were resolved on a 1%agarose denaturing gel and transferred to nitrocellulose. RNA blots werehybridized with ³²P labelled TGF-α riboprobes or exon 2 of human gastrinthat did not cross-hybridize with endogenous mouse gastrin mRNA.

[0063] Peptide radioimmunassay of Gastrin. Tissues were extracted andassayed for gastrin immunoreactivity by radioimmunoassay as describedpreviously using antibody 2604 which is specific for biologically activeC terminally amidated gastrin in a gastrin radioimmunoassay as describedin Rehfeld, J. F., Scand. J. Clin. Lab. Invest. 30:361-368 (1972).Tyrosine monoiodinated human gastrin 17 tracer was used in all assaysand synthetic human gastrin 17 was used as a standard.

[0064] Peptide Radioimmunoassay of TGF-α: Tissues were frozen in liquidnitrogen, ground to a powder with mortar and pestle, and subjected toacid-ethanol extraction as described in Todaro, G. J. et al, Proc. Natl.Acad Sci. (USA), 77:5258-5262 (1980). Extracts were reconstituted withwater, and protein concentrations determined with a Coomassie blue dyebinding assay (Bio-Rad Laboratories, Hercules, Calif.). Aliquots fromthe pancreata were tested in duplicate in a TGF-α radioimmunoassay,which measured competition with ¹²⁵I TGF-α for binding to a solid-phaserabbit antibody raised against the C-terminus of rat TGF-α (kit fromBioTope, Seattle, Wash.).

[0065] Blood Glucose. Blood glucose was determined either afterovernight fasting or after IPGTT by glucose oxidase method.

[0066] Tissue Insulin Analysis. At the end of each study, the animalswere sacrificed and pancreas removed. Small biopsies were taken fromseparate representative sites throughout the pancreas and immediatelysnap-frozen in liquid nitrogen for immunohistochemistry, protein, andinsulin determinations. Snap-frozen pancreatic samples (n=5) wererapidly thawed, disrupted ultrasonically in deionized water and aliquotstaken for protein determination and the homogenate subjected toacid/ethanol extraction prior to insulin determination by RIA.

[0067] Histological Analysis. The pancreata were removed, weighed,similarly oriented in cassettes, fixed in Bouin's solution and embeddedin paraffin by conventional procedures.

[0068] Tissue Preparation and Immunohistochemistry. Freshly excisedpancreata were dissected, cleared of fat and lymph nodes, fixed inBouin's fixative, and then embedded in paraffin for sectioning. Routinesections were stained with hematoxylin and eosin according to standardmethods. Pancreatic tissue from adult 17 week old MT-TGF-α (MT-42)transgenic mice were immunostained for insulin to examine the effect ofTGF-α over-expression on islet development. Insulin positive cells inTGF-α-induced metaplastic ductules were identified usingimmunoperoxidase staining guinea pig anti-human insulin sera (Linco,Eureka, Mo.); a pre-immune guinea pig serum was used as a control.Immunohistochemistry was performed on 5μ paraffin sections by theperoxidase/antiperoxidase method of Sternberger using a monoclonalrabbit antigastrin antibody. See, Sternberger, L. A.,Immunocytochemistry, 2^(nd) Ed. (1979) NY: Wiley. 104-170.

[0069] Point-Counting Morphometrics. The relative volume of islets,ducts, or interstitial cells was quantitated using the point-countingmethod described in Weibel, E. R., Lab Investig., 12:131-155 (1963). Ata magnification of 400×, starting at a random point at one corner of thesection, every other field was scored using a 25 point ocular grid. Anunbiased but systematic selection of fields was accomplished using themarkings of the stage micrometer. Intercepts over blood vessels, fat,ducts, lymph nodes, or interlobular space were subtracted to give thetotal pancreatic area. A minimum of 5000 points in 108 fields(systematically chosen using the stage micrometer) were counted in eachblock, with the relative islet volume being the number of interceptsover islet tissue divided by the number over pancreatic tissue. Theabsolute islet mass or islets was calculated as the relative isletvolume times pancreatic weight. See, Lee, H. C., et al, Endocrinology,124:1571-1575 (1989).

[0070] Statistical Analysis. Differences between means were compared forsignificant differences using the Student's t test for unpaired data.

Example 1 Assay For Insulin Production in TGF Transgenic Pancreas

[0071] Immunoperoxidase staining showed numerous insulin staining cellsin the metaplastic ducts from the TGF-α transgenic pancreas (FIG. 1A),whereas insulin staining cells were virtually absent from thenon-transgenic ducts (less than 6.1%). When at least 600 ductularcells/animal were scored at a final magnification of 400×, insulinpositive cells were seen at a frequency of 6.0+/−0.9% (n=5) in themetaplastic ductules of TGF-α transgenic mice. Occasional ductular cellsstained with the same intensity of insulin staining as the adjacentislets, but most had less intense staining (FIG. 1B). The low level ofinsulin staining of the ductular cells resembles that ofprotodifferentiated cells reported in the ducts of the developingpancreas. Pictet, R. and W. J. Rutter, Development of the embryonicendocrine pancreas, in Endocrinology, Handbook of Physiology, ed. R. O.Greep and E. B. Astwood (1972) American Physiological Society:Washington, D.C. 25-66; and Alpert, S. et al Cell, 53:295-308 (1988).

[0072] However, despite the increased number of insulin positive cellsin the metaplastic ducts, the islet mass of the TGF-α transgenic micewas not increased. The islet mass as quantitated by point countingmorphometrics was 2.14 mg+/−0.84 (mean+/−se, n=5) in the TGF-αtransgenic pancreas compared to 1.93 mg+/−0.46 (n=6) non transgeniclitter mates.

[0073] Thus, TGF-α over-expression alone did not effect transition ofthese protodifferentiated duct cells into fully differentiated islets.This implies that islet differentiation requires other factors absentfrom the adult pancreas of TGF-α transgenic mice. Since differentiationof protodifferentiated islet precursors occurs during late fetaldevelopment, factors regulating this transition would likely beexpressed in islets during this period. Among the factors expressed inthe developing islets are the gastrointestinal peptides, the gastrins.

Example 2 Pancreatic Gastrin Expression from the INSGAS Transgene

[0074] To examine the possible role of gastrin in regulating isletdifferentiation, transgenic mice were created that express a chimericinsulin promoter-gastrin (INSGAS) transgene in which the insulinpromoter directs pancreas specific expression of the gastrin transgene(FIG. 2A). Unlike the gastrin gene, insulin gene expression is notswitched off after birth. Thus, the INSGAS transgene results in apersistence of gastrin expression in the adult pancreas.

[0075] The INSGAS transgene comprised 370 bp of 5′ flanking DNA and thefirst non-coding exon of the rat insulin I gene. Cordell, B., et al,Cell 18:533-543 (1979). It was ligated to a BamH1-EcoR1 fragmentcontaining 1.5 kb intron 1 and exons 2 and 3 of the human gastrin genewhich encodes the preprogastin peptide precursor. Wiborg, O., et al,Proc. Natl. Acad Sci. USA, 81:1067-1069 (1984); and Ito et al Proc.Natl. Acad. Sci. USA, 81:4662-4666 (1984). A 4.8 kb INSGAS fragment wasisolated and microinjected into inbred FVB, one cell mouse embryos.Hogan, B. et al, Manipulating the mouse embroy: A laboratory manual,(1986) NY:Cold Spring Harbor.

[0076] Gastrin immunoreactivity in pancreatic and stomach extracts fromtransgenic and non-transgenic mice was assayed by radioimmunoassay usingantisera 2604 (Rehfeld, J., et al, Scand. J. Clin. Lab. Invest.,30:361-368 (1972)) specific for the bioactive amidated C-terminus ofgastrin.

[0077] Beta cell specific gastrin expression from the INSGAS transgenewas observed based on immunostaining of pancreatic tissues with agastrin monoclonal antibody.

[0078] Northern blots of RNA isolated from different tissues of 8 weekold INSGAS transgenic mice were hybridized with a human gastrin exon 2probe. High levels of gastrin transgene mRNA were seen in the pancreasbut not in any other tissues. This probe is specific for the humangastrin gene; no hybridization is seen in antral RNA of INSGAS andnon-transgenic FVB mice express high levels of murine gastrin mRNA.Radioimmunoassay of pancreatic extracts from INSGAS transgenic miceshowed high levels of gastrin immunoreactivity that exceed the gastrincontent in the gastric atrium expressed from the endogenous murine gene(FIG. 2B). No gastrin immunoreactivity was detected in pancreaticextracts of non-transgenic control mice (FIG. 2B). The gastrinradioimmunoassay is specific for carboxy amidated precursors, indicatingthat the gastrin peptide precursor is efficiently processedpost-translationally to the bioactive peptide. Immunohistochemistry witha gastrin monoclonal antibody shows pancreatic beta islet cell specificexpression of gastrin.

[0079] Although the INSGAS transgenic mice had high expression ofgastrin in the postnatal pancreas (FIG. 2B), the INSGAS transgenic micehad pancreatic histology identical to controls. Islet mass asquantitated by point-counting morphometrics (Weibel, E. R., LabInvestig. 12:131-155 (1963)) was identical in 5-6 week old INSGAS mice(1.78+/−0.21 mg, n=1) and age matched non-transgenic controls(1.74+/−0.18 mg, n=11). Thus, sustained expression of gastrin in thepostnatal pancreas alone does not stimulate islet cell growth.

Example 3 Histological Examination of TGF-α and TGF-α/INSGAS Pancreas

[0080] Stimulation of islet growth by gastrin may require stimulation byother growth factors to create a responsive population of cells.Therefore, effects of gastrin stimulation were studied in TGF-αtransgenic mice which have metaplastic ducts that contain insulinexpressing cells resembling protodifferentiated islet-precursors. Toassess the interaction between gastrin and TGF-α, three groups of micewere bred with equivalent FVB/CD1 strain genetic backgrounds:non-transgenic control, TGF-α single transgenic and INSGAS/TGF-α doubletransgenics. All three groups of mice were placed on 50 mM ZnCl₂ at 3weeks of age. At 17 weeks of age, the animals were sacrificed and thepancreas removed for histological evaluation. The pancreas from TGF-αand INSGAS/TGF-α mice had similar gross morphological appearances:resilient, firm and compact in contrast to the soft diffuse controlpancreas. TGF-α expression was equivalent in TGF-α and INSGAS/TGF-αgroups when measured by Northern blot analysis (data not shown) and byradioimmunoassay. The pancreatic TGF-α immunoreactive peptide levelswere 12.2+/−1 and 18.9+/−8 ng/mg protein (Mean+/−SD) in the TGF-α andINSGAS/TGF-α mice, respectively.

[0081] Light micrographs of hematoxylin stained paraffin sections ofpancreas from the three groups of mice studied (A: INSGAS/TGF-α; B:FVB/CD1 controls; and C: TGF-α) were made. The INSGAS/TGF-α pancreas hadsome areas of increased ductular complexes and slightly increasedinterstitial cellularity; the field shown (FIG. 3A) had the mostseverely abnormal morphology seen in the five animals; most of thepancreas was indistinguishable from controls (FIG. 3B). In contrast, thefield of TGF-α pancreas (FIG. 3C) was typical and showed theinterstitial cellularity and fibrosis combined with florid ductularmetaplasia described by Jhappan et al, supra.

[0082] Pancreatic gastrin synergistically interacts with TGF-α toincrease islet mass and inhibit the ductular metaplasia induced by TGF-αover-expression. Mating the homozygous MT-TGF-α (MT-42) mice (TGF-α)with heterozygotic INSGAS mice gave offspring that were eitherheterozygotic TGF-α single transgenic or double transgenic containingboth INSGAS and TGF-α transgenes (INSGAS/TGF-α). Since INSGAS were FVBstrain and TGF-α were CD1 strain, TGF-α homozygotes and CD1 controls(CON) were both mated with FVB to produce FVB/CD1 strain background forall three groups of mice. Mice were treated with 50 mM ZnCl₂ from 3weeks until sacrifice at age 17 weeks. The pancreas was removed,weighed, similarly oriented in cassettes, fixed in Bouin's solution andembedded in paraffin. One random section from each animal was used toquantitate the relative volumes of ductules and islets by point-countingmorphometrics (Weibel, E. R., Lab Investig., 12:131-155 (1963)). Atleast 2000 points over tissue were counted as intercepts of a 50 pointgrid at 170× magnification; the entire section was covered withoutoverlap. The mass of ductules or islets was calculated by multiplyingthe relative volume and the animal's pancreatic weight.

[0083] To normalize different mean body weights, the mass was expressedas μg/g body weight. Results are mean and standard errors for 5-6animals in each group as determined by Student's t test (p<0.05).

[0084] Expression of gastrin from the INSGAS transgene reduced theductular metaplasia caused by TGF-α over-expression. At 17 weeks, thepancreatic histology of the INSGAS/TGF-α mice (FIG. 3A) resembled thatof the control pancreas (FIG. 3B) more than that of the TGF-α mice (FIG.3C).

[0085] This was confirmed by quantitating pancreatic ductular mass inthe TGF-α and INSGAS/TGF-α transgenic mice and the FVB/CD1 controls bypoint-counting morphometrics (FIG. 4A). Co-expression of gastrin andTGF-α in the INSGAS/TGF-α pancreas also significantly increased theislet mass compared to controls (FIG. 4B), whereas islet mass was notincreased by expression of the TGF-α or gastrin transgenes alone. Theblood glucose concentration was not significantly different among thethree groups of mice.

Example 4 Effects of TGF-α and Gastrin on Pancreatic Insulin Content inNormal Rats

[0086] This experiment was designed to study the effects on pancreaticinsulin content in non-diabetic animals treated with TGF-α, a gastrin,or a combination of TGF-α and a gastrin as compared to control animals(untreated). Groups (n=5) of normal Wistar rats were assigned to one ofthe following four treatment groups.

[0087] Group I: TGF-α: recombinant Human TGF-α was reconstituted insterile saline containing 0.1% BSA and was administered i.p. at a doseof 0.8 μg/day for 10 days.

[0088] Group II: Gastrin: synthetic Rat Gastrin 1 was dissolved in verydilute ammonium hydroxide and reconstituted in sterile saline containing0.1% BSA. It was administered i.p. at a dose of 0.8 μg/day for 10 days.

[0089] Group III: TGF-α+Gastrin: a combination of the above preparationswas administered i.p. at the dose levels given above for 10 days.

[0090] Group IV: Control animals received an i.p. injection of vehiclealone for 10 days.

[0091] At the end of the study period (10 days), all animals weresacrificed and samples of pancreas taken as follows: five biopsyspecimens (1-2 mg) of pancreatic tissue were taken from separaterepresentative sites in each rat pancreas and immediately snap frozen inliquid nitrogen for analysis of insulin content. For analysis ofpancreatic insulin content, the snap frozen pancreatic samples wererapidly thawed, disrupted ultrasonically in distilled water and aliquotstaken for protein determination and acid/ethanol extraction prior toinsulin radioimmunoassay (Green et al, (1983) Diabetes 32:685-690).Pancreatic insulin content values were corrected according to proteincontent and finally expressed as μg insulin/mg pancreatic protein. Allvalues calculated as mean+/−SEM and statistical significance evaluatedusing Student's 2-sample t-test. TABLE 1 Treatment of Normal Rats withTGF-α and Gastrin Pancreatic Insulin Content Treatment (μg insulin/mgprotein) Control 20.6 +/− 6.0   TGF-α 30.4 +/− 7.4*  Gastrin 51.4 +/−14.0** TGF-α + Gastrin 60.6 +/− 8.7***

[0092] As shown in Table 1, above, pancreatic insulin content wassignificantly increased (p=0.007) in the TGF-α+gastrin treated animalsas compared to control animals; there was an approximately three-foldincrease in pancreatic insulin content as compared to control animals.These data support the hypothesis that the combination of TFG-α andgastrin does produce an increase in the functional islet β-cell volume.This increase reflects an overall condition of β-cell hyperplasia(increase in number) rather than β-cell hypertrophy (increase in size ofindividual β-cells).

Example 5 Effect of Combination of TGF-α and Gastrin on PancreaticInsulin Content in Diabetic Animals

[0093] The second experiment was designed to determine whether thecombination of TGF-α and gastrin could increase pancreatic insulincontent in diabetic animals (streptozotocin (STZ) treated) to levelscomparable to those in normal (non-STZ treated) animals.

[0094] Normal Wistar rats received a single iv injection of STZ at adose of 80 mg/Kg body weight. This dose of STZ was intended to ensurethat the study animals were rendered diabetic but that they retained afunctioning but reduced β-cell mass. The STZ was dissolved immediatelybefore administration in ice-cold 10 mM citric acid buffer. The animalswere monitored daily; persistent diabetes was indicated by glycosuriaand confirmed by non-fasting blood glucose determinations. One weekafter induction of diabetes, rats were randomly allocated into twogroups (n=6) as follows. Group I: TGF-α + Gastrin: STZ diabetic ratswere treated with a single i.p. injection of a combination ofrecombinant human TGF-α and synthetic rat Gastrin 1; both preparationswere administer- ed at a dose of 0.8 μg/day for 10 days. Group II:Control: STZ diabetic rats received an i.p. injection of vehicle alonefor 10 days.

[0095] At the end of the study period, all animals were sacrificed andsamples of pancreas taken and analyzed as described in Example 4 and theresults are given in Table 2. TABLE 2 Treatment of Streptozotocin Ratswith TGF-α and Gastrin Pancreatic Insulin Content Treatment (μgInsulin/mg protein) Control (STZ alone) 6.06 +/− 2.1 STZ plus TGF-α +Gastrin 26.7 +/− 8.9

[0096] The induction of diabetes by STZ was successful and produced amoderate but sustained degree of hyperglycemia. Total insulinopaenia wasnot sought so as to ensure that the study animals retained afunctioning, but reduced β-cell mass.

[0097] As shown in Table 2, above, the pancreatic insulin content of thecontrol streptozotocin treated animals was less than one third that ofnormal rats (20.6±6.0 mg insulin/mg protein, see Table 1 above) as aresult of destruction of β-cells by the STZ. In STZ animals treated witha combination of TGF-α and gastrin, the pancreatic insulin content wasmore than four-fold that of the animals which received STZ alone, andstatistically the same as that of normal rats.

[0098] Diabetes mellitus is a disease in which the underlyingphysiological defect is a deficiency of β-cells as a result either ofdestruction of the β-cells due to auto-immune processes or of exhaustionof the potential for the β-cells to divide due to chronic stimulationfrom high circulating levels of glucose. The latter eventually leads toa situation when the process of β-cell renewal and/or replacement iscompromised to the extent that there is an overall loss of β-cells and aconcomitant decrease in the insulin content of the pancreas. The aboveresults demonstrate that a combination of TGF-α and gastrin can be usedto treat diabetes by stimulating the production of mature β-cells torestore the insulin content of the pancreas to non-diabetic levels.

Example 6 Effects of TGF-α and Gastrin on IPGTT in STZ-Induced DiabeticAnimals

[0099] Two groups (average body weight 103 g) of STZ induced diabeticWistar rats (n=6/group) were treated for 10 days with a daily i.p.injection of either a combination of TGF-α and gastrin or PBS. Fastingblood glucose was determined for all rats on days 0, 6, and 10. In orderto establish that this insulin was secreted and functional, IPGTT testswere performed. At day 10, intraperitoneal glucose tolerance tests(IPGTT) were performed following an overnight fast. Blood samples wereobtained from the tail vein, before and 30, 60 and 120 minutes afteradministration of an i.p. glucose injection at a dose of 2 g/kg bodyweight. Blood glucose determinations were performed as above. The bloodglucose levels were similar in both study groups at time 0 but the TFGαand gastrin treated rats demonstrated a 50% reduction in blood glucosevalues (see FIG. 5), as compared to control rats at 30, 60, and 120 min.following the i.p. glucose load.

Example 7 Effects of TGF-α and Gastrin on Body Weight Gain and InsulinContent in Diabetes Prone Animals

[0100] Zucker rats were obtained at 30 days of age approximately 10-15days prior to development of obesity. Besides the diabetes prone Zuckerrats (genotype fa/fa, autosomal recessive mutation for obesity anddiabetes), lean non-diabetic littermates (genotype +/+) also wereincluded in the study as described below. The rats were monitored dailyfor development of obesity and diabetes by determining body weight andblood glucose. The onset of diabetes in Zucker rats usually startedbetween days 45-50 and was confirmed by a significant increase in bloodglucose levels, as compared to the levels in age-matched lean controls.

[0101] The study included 5 groups of 5 rats each as described in Table3. Groups 1 and 2 (lean, non-diabetic) were treated with a TGF-α andgastrin combination or PBS respectively from day 0 to day 10. Groups 3,4 and 5 included obese, early diabetic Zucker rats, genotype fa/fa.Group 3 received a combination pretreatment for 15 days (day −15 to day0) prior to onset of diabetes and continuing post onset of diabetes for10 additional days (day 0 to day 10). Group 4 was treated with acombination of TGF-α and gastrin for 10 days after onset of diabetes andGroup 5 was treated with PBS over the same time period. At the end ofthe study, the rats were sacrificed and the pancreas removed. Smallbiopsies were taken from separate representative sites for protein andinsulin determinations as described above.

[0102] The body weight gain in obese diabetic Zucker rats withpretreatment, treatment only or with saline (groups 3, 4, and 5 in Table3) did not show any significant differences among the groups. It isinteresting to note that even prolonged treatment (25 days, group 3)with TGF-α+gastrin was without effect on normal weight gain. Withinerror limits body weight gain was identical in all the groups.

[0103] The effect of TGF-α+gastrin treatment on fasting blood glucose inthe obese Zucker rats was compared to the corresponding PBS controls.Fasting blood glucose was first significantly increased by day 15(4.0±0.6 vs. 5.0±0.2) and this time point was chosen as the startingtime for the 10-day treatment period with TGF-α +gastrin or with PBScontrol. Fasting blood glucose levels were not significantly altered bythe TGF-α +gastrin treatment or by PBS. Fasting blood glucose valueswere lower in lean, as compared to obese animals whether or not theywere treated with the growth factors or with PBS. TABLE 3 Pretreatment ±Body Wt Group Geotype Condition Treatment (days) PBS Control Gain (% ±SE) 1. +/+ lean, non-diabetic None Yes 117 ± 2.1 2. +/+ lean,non-diabetic  0 + 10 No 119 ± 1.9 3. fa/fa obese, early diabetic −15 +10   No 202 ± 15  4. fa/fa obese, early diabetic  0 + 10 No 119 ± 1.0 5.fa/fa obese, early diabetic None Yes 129 ± 1.3

[0104] The results of treatment with TGF-α and gastrin in the Zucker ratmodel of Type 2 diabetes showed no significant differences in bloodglucose levels between the treatment and control groups, probablyreflecting the transient hypoglycemic effect following a prolongedperiod (18 hrs) of fasting. The immunohistochemical studies revealedsignificant increases in the number of single foci of insulin containingcells in the TGF-α and gastrin treated animals, as compared to controlanimals. These findings demonstrated an increase in single β-cells inadult rat pancreas following treatment with TGF-α and gastrin.Interestingly, such single β-cell foci are not commonly seen in adult(unstimulated) rat pancreas. These findings support a therapeutic rolefor TGF-α and gastrin in Type 1 and Type 2 diabetes since treatment istargeted at both β-cell neogenesis and replication.

[0105] The present invention is based in part on studies whichdemonstrated numerous insulin staining cells in the TGF-α-inducedmetaplastic ductules. The low level of exocrine and endocrine geneexpression in the metaplastic ductal cells resembled that ofprotodifferentiated ductal cells seen in the early stage of fetalpancreatic development. Formation of islets (neogenesis) results fromproliferation and differentiation of these protodifferentiated insulinexpressing cells. Histologically this is manifested as islets appearingto bud from the pancreatic ducts (nesidioblastosis). In the MT-42 TGF-αtransgenic mice, ductular metaplasia was not seen in the immediatepost-natal period, but only at 4 weeks of age. This indicates that TGF-αover-expression induced insulin expression in duct epithelia rather thanprolonging the persistence of islet precursors found in fetal pancreaticducts. Although the metaplastic ductules contained numerous insulinpositive cells, the islet mass of the TGF-α transgenic mice was notincreased over controls. The studies reported above demonstrate thatcomplete islet cell neogenesis is reactivated in vivo in mammals in theductular epithelium of the adult pancreas by stimulation with agastrin/CCK receptor ligand, such as gastrin, and/or an EGF receptorligand, such as TGF-α. Studies are reported on the transgenicover-expression of TGF-α and gastrin in the pancreas which elucidate therole of pancreatic gastrin expression in islet development and indicatethat TGF-α and gastrin each play a role in regulating islet development.Thus, regenerative differentiation of residual pluripotent pancreaticductal cells into mature insulin-secreting cells is a viable method forthe treatment of diabetes mellitus, by therapeutic administration ofthis combination of factors or compositions which provide for their insitu expression within the pancreas.

[0106] The present invention is not limited by the specific embodimentsdescribed herein. Modifications that become apparent from the foregoingdescription and accompanying figures fall within the scope of theclaims.

[0107] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entirety.

1-18 (canceled)
 19. A method for treating diabetes mellitus in anindividual in need thereof, said method comprising: administering tosaid individual a composition providing a gastrin/CCK receptor ligandthat stimulates islet cell neogenesis in an amount sufficient to effectdifferentiation of pancreatic islet precursor cells to matureinsulin-secreting cells.
 20. The method according to claim 19, whereinsaid ligand is a gastrin.
 21. The method according to claim 20, whereinsaid gastrin is selected from the group consisting of gastrin 34,gastrin 17, and gastrin
 8. 22. The method according to claim 19, whereinsaid ligand is a cholecystokinin.
 23. The method according to claim 22,wherein said cholecystokinin is selected from the group consisting ofCCK 58, CCK 33, CCK 22, CCK 12 and CCK
 8. 24. The method according toclaim 19, wherein said islet cell neogenesis is further stimulated whensaid ligand is administered in combination with an EGF receptor ligand.25. The method according to claim 20, wherein said ligand is an activeanalog of gastrin, an active fragment of gastrin or a modified gastrin.26. A method for treating diabetes in a patient in need thereof, saidmethod comprising the step of: ransplanting into said patient pancreaticislets which have been provided ex vivo with a sufficient amount of agastrin/CCK receptor ligand to induce differentiation of precursor cellsin said islets to mature insulin-secreting β-cells.
 27. The methodaccording to claim 26, wherein the number of β-cells in said islets hasbeen expanded prior to said transplanting step by providing saidpancreatic islets with a sufficient amount of an EGF receptor ligand toincrease the number of β-cells in said islets.
 28. The method accordingto claim 19 or claim 27, wherein said diabetes is Type 2 diabetes.
 29. Amethod for enhancing production of mature insulin-secreting cells, saidmethod comprising: providing to a population of precursor cells aneffective amount of a gastrin/CCK receptor ligand to effectdifferentiation of said precursor cells to mature insulin-secretingcells.
 30. A composition comprising: pancreatic β cells, wherein saidculture is obtained by providing precursor cells of said pancreatic βcells with a sufficient amount of a gastrin receptor agonist to inducedifferentiation of said precursor cells to mature pancreatic β cells.31. The method according to claim 19, wherein said composition isadministered systemically.
 32. A method for stimulating pancreatic isletcell neogenesis in an individual in need thereof, said methodcomprising: administering to said individual a composition comprising agastrin in an amount sufficient to effect differentiation of pancreaticislet precursor cells to mature insulin-secreting islet cells, whereinsaid composition is administered systemically.
 33. The method accordingto claim 19, wherein said gastrin/CCK receptor ligand is a proteinaceousreceptor ligand.
 34. A kit comprising: one or more containers filledwith one or more ingredient for a pharmaceutical composition comprisinga gastrin receptor ligand.
 35. The kit according to claim 34, whereinsaid one or more ingredient are a gastrin and a pharmaceutical carrier.36. The kit according to claim 34, wherein said one or more ingredientare lyophilized.